Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Hemophilia is an X-linked hemorrhagic disorder resulting from mutations in either the FVIII (hemophilia A) or FIX (hemophilia B) genes, with an incidence of approximately 1:5,000 male individuals worldwide. Affected individuals commonly present spontaneous hemorrhages and prolonged bleeding after trauma or surgery. Severely affected subjects present FVIII or FIX levels lower than 1% of normal and comprise the majority of clinically symptomatic cases. The remaining patients have mild to moderate disease with factor levels of 1-30%[1].
The clinical presentation of hemophilia is essentially indistinguishable for FVIII or FIX deficiency. However, there is clear evidence that the clinical phenotype of hemophilia varies among patients with similar residual factor levels or even with the same underlying mutation [2-4]. Therefore, it is possible that other genetic or acquired factors influence the hemophilia phenotype. The current understanding of the genetic basis of venous thrombosis provides an opportunity to determine whether these risk factors could improve the hemophilia phenotype.
Thrombin generation is in part controlled by activated protein C (APC), which is formed by limited proteolysis of the zymogen protein C by the thrombin-thrombomodulin complex. The anticoagulant effect of APC results from the inactivation of both factors Va and VIIIa on membrane surfaces[5]. The most common inherited thrombophilia results from a mutation in the FV gene (Arg 506 to Gln) known as FV Leiden (FVL). Because Arg 506 is the initial cleavage site for APC, FVL is inactivated at approximately one tenth the rate of normal FVa [6], which result in high thrombin levels that create a procoagulant state.
FVL is the most commonly investigated modifier of the hemophilia phenotype because it is present in 2-5% of the Caucasian population [7, 8]. Initial reports suggested the amelioration of the severe hemophilia A phenotype among subjects with FVL [9, 10]. Further studies, however, failed to demonstrate the clinical impact of such association. In screening over 700 hemophilia subjects, 35 cases of FVL have been identified. In only half of these cases (14 hemophilia A and 1 hemophilia B) the association was considered beneficial in terms of frequency of bleeds and/or factor consumption over time [9-14]. The reasons for these discrepancies are not clear but could relate to the small number of subjects, differences in age groups, the presence of underlying infectious diseases, and the retrospective nature of the study. The results of a pediatric study have been informative in this matter since many of the complications common among adults are not confounding factors in children. This case-control study demonstrated that among hemophilia A children with FVL or with other thrombophilia risk factors, the onset of the first bleeding episode was delayed [14].
There is also in vitro evidence that the FVL mutation can modify thrombin generation in FVIII [15] or FIX deficient plasma [16]. Moreover, the assessment of the fibrinolytic system in hemophilia revealed that thrombin-induced clots in FVIII or FIX deficient plasma were lysed prematurely [17]. Therefore, the enhanced generation of thrombin by FVL may also increase the resistance of the fibrin clot to premature lysis [18, 19].